A. HIV functional cure strategy based on novel chimeric antigen receptors (CARs). Durable (life-long?) HIV suppression requires the CAR to display not only high potency, but also to approach the ideals of inescapabilty and non-immunogenicity. We have designed bispecific CARs containing CD4 linked to a second moiety that binds to a distinct highly conserved region of Env. The second moiety enhances potency, and also prevents the CD4 from acting as an HIV entry receptor. Particular success was achieved with the carbohydrate binding domain of human mannose binding lectin (MBL), which binds to the universally conserved high-mannose patch on gp120. The CD4-MBL bispecific CAR shows greatly enhanced potency compared to the monospecific CD4 CAR, with wide breadth for genetically diverse HIV-1 isolates; moreover, the lectin moiety prevents the CD4 from rendering CAR-expressing CD8+ T cells susceptible to infection (Ghanem et al. 2018). For NHP studies (in collaboration with Dr. Pamela Skinner, U. Minnesota and Dr. Elizabeth Connick, U. Arizona), we generated an all-rhesus variant of CD4-MBL for co-expression with CXCR5 to facilitate homing of CAR-T cells to B cell follicles where infected Tfh cells serve as major sites of HIV persistence. CXCR5 was found to promote selective migration of CAR-T cells toward the chemokine ligand CXCL13 in vitro, as well their accumulation inside B cell follicles in rhesus lymphoid tissue explants ex vivo (Haran et al. Front. Immunol. 2018). When injected into a rhesus macaque, CTV labeled CAR/CXCR5 T cells were found in various tissues including spleen and lymph nodes. Newly generated CAR constructs co-expressing fluorescent reporter genes, or containing epitope tags in the CAR ectodomain, will facilitate studies of follicular trafficking of CAR-T cells in vivo, and their ability to deplete infected Tfh cells. To further enhance potency of CD4-based CARs, we have studied additional polypeptide sequences for the second moiety derived from alternative Env-binding human proteins. For the new CARs analyzed, the second moieties significantly enhanced potency. Initial experiments suggest that these new CD4-based CARs not only avoid entry receptor activity on CD8+ T cells, but also block endogenous CD4-mediated infection of CAR-expressing CD4+ T cells. Future efforts will focus on further analysis of the new CD4-based bispecific CARs, as well as trispecific constructs. We have initiated collaborative studies (Dr. Thor Wagner, Seattle Childrens) to directly compare the CD4-based CARs with scFv-containing CARs derived from several broadly neutralizing monoclonal antibodies. In addition to our continuing NHP studies, we collaborating in a humanized mouse model (Dr. Brad Jones, Weill/Cornell Med and Dr. Catherine Bollard, Childrens National DC), to test CAR-T cells wherein the CAR is introduced into virus-specific expanded T cells derived from infected subjects. The goal is to test whether the CAR can significantly enhance the potency of expanded HIV-specific T cells, and/or confer potent anti-HIV activity to expanded T cells against other common viral pathogens (e.g. CMV). B. HIV gp120 locked in the pre-CD4 conformation as novel vaccine immunogen. Our collaborative studies with Dr. Paolo Lusso (NIAID) led to the design of a novel Env trimer constructs (soluble and full-length membrane-associated forms) locked in the pre-fusion state by structure guided introduction of new disulfide bonds connecting the inner and outer domains (Zhang et al, 2018). This interdomain stabilization strongly impaired CD4 binding and function. Importantly, the locked trimers displayed enhanced binding to broadly neutralizing antibodies and loss of reactivity with CD4-induced antibodies. Immunization of rabbits resulted elicited antibodies that neutralized tier-2 viruses, suggesting the value of this strategy for Env vaccine strategies aimed at neutralizing antibody.